This is a competitive renewal application to investigate the molecular mechanisms of the chemopreventive effect on cancer by tea polyphenols. During the previous funding period, we investigated the effect of tea polyphenols on AP-1 and NF(kappa)B signal transduction pathways in cell culture and transgenic mouse models. In JB6 cells, Ras transformed 30.7b cells and keratinocytes, tea polyphenol(-)-epigallocatechin-3-gallate (EGCG) and theaflavins were shown to inhibit tumor promoter-induced or oncogene-induced AP-1 and NF(kappa)B activation in a concentration-dependent manner. The dose range of these tea polyphenols that effectively inhibited AP-1 or NF(kappa)B activity is similar to that which can inhibit cell transformation and skin carcinogenesis. We further demonstrated that EGCG and theaflavins inhibit tumor promoter-induced activation of protein kinases, which are upstream of AP-1, NF(kappa)B and other transcription factors. Tea polyphenols are known to have a high affinity for binding to proline-rich proteins and many protein kinases in tumor promoter-induced signal transduction pathways contain proline-rich regions. Therefore, we hypothesized in our renewal application that the chemopreventive effect of the tea polyphenol EGCG and its derivatives is derived from their inhibition of the activation of protein kinases in signal transduction pathways and the inhibition occurs through their interaction with proline-rich regions of these proteins. We will use 3H-EGCG, newly synthesized EGCG derivatives, cell culture and protein analysis to examine the anti-tumor promotion activity of tea polyphenols. The specific aims to address the hypothesis are: (1) to investigate the role of proline-rich protein kinases (e.g., MEK1/2, JNKs, and PI-3 kinase) in tumor promoter-induced cell transformation in vitro; (2) to investigate whether protein kinases with proline-rich regions are involved in the inhibition of cell transformation by tea polyphenols; (3) to investigate whether inhibition of the activity of JNKs, MEKs, or PI-3 kinase by tea polyphenols occurs through the direct targeting of these proline-rich kinases; and (4) to investigate the structure-effect relationship of EGCG derivatives in the inhibition of the tumor promoter-induced action of proline-rich protein kinases and cell transformation. We have established experimental conditions through preliminary investigations and expect that the proposed molecular mechanism studies will lead to the development of better chemopreventive agents and facilitate the design of more effective chemoprevention trials.